Fusion protein between human macif and a heterologous pi anchor domain

ABSTRACT

Fusion proteins comprising the extracellular domain of the human MACIF (Membrane Attack Complex Inhibition Factor) gene product and a heterologous phosphatidylinositol (PI) anchor domain are provided.

This application is a continuation of application Ser. No. 08/021,724filed Feb. 22, 1993, now abandoned, which is in turn a division ofapplication Ser. No. 07/510,610, filed Apr. 18, 1990, noe abandoned.

FIELD OF THE INVENTION

This invention relates to genes coding for a protein having human MACIF(membrane attack complex inhibition factor) activity, expression vectorswith the genes respectively inserted therein, cells transformed with theexpression vectors, and proteins having human MACIF activity. The term"protein having human MACIF activity" includes, within the meaningthereof, a group of proteins which regulate the complement system in thefinal stage of complement activation and inhibit damaging of human cellsand tissues as a result of membrane attack complex formation.

BACKGROUND OF THE INVENTION

The present inventors previously succeeded in isolating a naturallyoccurring human MACIF, a so-far unknown protein regulating thecomplement system, in pure form from the human normal erythrocytemembrane (Japanese Patent Application No. 63-310642). They found thatthe human MACIF inhibits the activation of the late complementcomponents or, in other words, inhibits hemolysis resulting from humanMAC formation and, in this respect, the human MACIF is distinguishedfrom and superior to the known complement-regulating substances thatinhibit the activation of the early complement components, and that thenaturally occurring human MACIF has the following N-terminal amino acidsequence: ##STR1## Furthermore, they found that this naturally occurringhuman MACIF is a glycoprotein having a molecular weight of 18,000±1,000(as determined by SDS-polyacrylamide gel electrophoresis) with aphosphatidylinositol anchor (hereinafter abbreviated as "PI-anchor") atthe C terminus.

For further studying of the above-mentioned protein having human MACIFactivity and for developing the practical use of the protein as a drug,it is essential to obtain the protein in a pure and homogeneous form,and in sufficiently large quantities. For this purpose, application ofthe recombinant DNA technology appears to be the most effective means.However, the gene required for the means has not been isolated as yet.

Accordingly, an object of the invention is to provide a gene coding fora protein having human MACIF activity.

Another object of the invention is to provide a replicable expressionvector capable of expressing the gene coding for a protein having humanMACIF activity.

A further object of the invention is to provide a microorganism or cellstransformed with the expression vector.

A still further object is to provide a genetically engineered proteinhaving human MACIF activity.

SUMMARY OF THE INVENTION

The present invention provides genes respectively coding forpolypeptides having human MACIF activity in which an amino acid sequenceis represented by the following general formula (I): ##STR2## In theabove formula, X and Y are defined as follows: X is H,

Met, or the amino acid sequence:

Met Gly Ile Gln Gly Gly Ser Val Leu Phe Gly Leu Leu Leu Val Leu Ala ValPhe Cys His Ser Gly His Ser;

Y is OH,

the amino acid sequence: ##STR3## or an amino acid sequence derived fromthis amino acid sequence by deleting therefrom one to thirty-two aminoacid residues from the C terminus thereof.

The present invention further provides expression vectors respectivelycontaining the genes mentioned above, microorganisms or cellstransformed with the vectors, and genetically engineered proteinsexpressed in the microorganisms or cells and having human MACIFactivity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the gene isolated from human leukocytes and codingfor an amino acid sequence including the naturally occurring humanMACIF.

FIG. 2 illustrates an amino acid sequence deduced from the nucleotidesequence of the gene as shown in FIG. 1.

FIG. 3 illustrates the whole structure of an expression vector,pKK223-3, for use in Escherichia coli.

FIG. 4 illustrates the whole structure of an expression vector, pVY1,for use in CHO cells.

FIG. 5 illustrates the results of a test of some translation productsfor their antihemolytic activity in guinea pig erythrocytes.

FIG. 6 illustrates the dose dependency of the inhibitory activity of atranslation product in hemolysis of guinea pig erythrocytes and theresult of neutralization of the product with an anti-human MACIFantibody.

FIG. 7 illustrates a construction scheme for the expression vector pVY1.

FIGS. 8a, 8b, 8c illustrates flow cytometric analysis of recombinanthuman MACIF expression in transformant CHO cells.

FIG. 9 illustrates the dose dependency of the inhibitory activity inhemolysis of guinea pig erythrocytes, of recombinant human MACIFexpressed in transfomant CHO cells and the result of neutralizationthereof with an anti-human MACIF antibody.

FIG. 10 illustrates the whole structure of a vector, pTEJ001, forexpression in Escherichia coli.

FIG. 11 illustrates the results of purification of a modified humanMACIF protein (E103) expressed in Escherichia coli using Q-Sepharose.

FIG. 12 illustrates the results of SDS-PAGE analysis of fractionsobtained by purification of the modified human MACIF protein E103expressed in Escherichia coli using Q-Sepharose.

FIG. 13 illustrates the dose dependency of the inhibitory activity inhemolysis of guinea pig erythrocytes of the modified human MACIF proteinE103 expressed in Escherichia coli and the result of neutralizationthereof with an anti-human MACIF antibody.

FIG. 14 illustrates the results of purification of a modified humanMACIF protein (E86) expressed in Escherichia coli using Q-Sepharose.

FIG. 15 illustrates the results of SDS-PAGE analysis of fractionsobtained by purification of the modified human MACIF protein E86expressed in Escherichia coli using Q-Sepharose.

FIG. 16 illustrates the dose dependency of the inhibitory activity inhemolysis of guinea pig erythrocytes of the modified human MACIF proteinE86 expressed in Escherichia coli and the result of neutralizationthereof with an anti-human MACIF antibody.

FIG. 17 illustrates the dose dependency of the inhibitory activity inhemolysis of guinea pig erythrocytes of the modified human MACIF protein(C77) expressed in transformant CHO cells and the results of aneutralization test thereof with an anti-human MACIF antibody.

FIG. 18 illustrates the dose dependency of the inhibitory activity inhemolysis of guinea pig erythrocytes of the modified human MACIF protein(C76) expressed in transformant CHO cells.

FIG. 19 illustrates the dose dependency of the inhibitory activity inhemolysis of guinea pig erythrocytes of the modified human MACIF protein(C70) expressed in transformant CHO cells.

DETAILED DESCRIPTION OF THE INVENTION

An example of the genes isolated from human leukocytes by the presentinventors is the gene defined by the DNA sequence shown in FIG. 1. Thisgene contains a gene sequence coding for the amino acid sequence of thatprotein which exhibits physiological activities of naturally occurringhuman MACIF and, in addition, a gene coding for a naturally occurringhuman MACIF precursor amino acid sequence (inclusive of a secretorysignal peptide and a PI anchor-attachment signal amino acid sequence,among others).

The secretory signal peptide-encoding gene is a gene coding for apeptide necessary for the protein having human MACIF activity which isproduced in host cells to be released from the cell, while the PIanchor-attachment signal amino acid sequence-encoding gene is a genecoding for a hydrophobic amino acid sequence portion necessary for theattachment of the PI anchor to the protein having human MACIF activity.

Based on C-terminal analysis of a purified form of the naturallyoccurring human MACIF, the present inventors found that, in thenaturally occurring human MACIF, the PI anchor is attached to the 76thamino acid (glutamic acid) residue of the amino acid sequence shown inFIG. 2. The PI anchor has a skeletal structure composed ofphospho-ethanolamine, glycan and phosphatidylinositol (PI). The aminogroup of phospho-ethanolamine is attached to the carboxyl group of theC-terminal amino acid residue of peptides, and the fatty acid sidechains of the PI is bound to the cell membrane. In this way, thePI-anchor serves as an anchor for binding peptides to the cell membrane.It was found that, in the amino acid sequence shown in FIG. 2, thesequence from the methionine residue at position -25 to the serineresidue at position -1 is the secretory signal sequence and that thesequence from the asparagine residue at position 77 to the prolineresidue at position 103 is the PI anchor-attachment signal sequence.Thus, the amino acid sequence comprising the 1st to 76th amino acidresidues constitutes a peptide core of naturally occurring human MACIF.In this specification, the term "human MACIF" means any proteinessentially composed of the peptide comprising the first amino acid(leucine) residue to the 76th amino acid (glutamic acid) residue and thePI anchor attached to the C terminus of the peptide. This human MACIFmay have a sugar chain of various kinds of structures depending on thehost cells used for phenotypic expression or depending on culturingconditions of cells used therein. Human MACIF with a sugar chain of anystructure as well as human MACIF without any sugar chain falls withinthe scope of the human MACIF according to the present invention.

For practical use as a drug having physiological activities of humanMACIF, a protein is not required to have all the constituent elements ofthe above-mentioned human MACIF. It may be lacking in the PI anchor, orit may partially differ in amino acid sequence from human MACIF,provided that it has human MACIF activity. Thus, the desired proteinsaccording to the present invention also include proteins having humanMACIF-like physiological activities but differing from the very humanMACIF in that a part of the amino acid sequence of human MACIF ismissing in them or replaced with some other amino acid sequence, in thatsome amino acid sequence is added to or inserted in them, in that theyhave no PI anchor and/or in that they have no carbohydrate chain ordiffer in the kind of carbohydrate. In this specification, theseobjective proteins are referred to as "modified human MACIF proteins".

The genes provided by the present invention include genes coding for theabove-mentioned human MACIF and modified human MACIF proteins. Astypical examples of the modified human MACIF protein-encoding genes,there may be mentioned a gene coding for the amino acid sequence fromthe 1st amino acid residue (Leu) to the 70th amino acid residue (Asn) ofthe above-mentioned amino acid sequence of formula (I), a gene codingfor the amino acid sequence from the 1st to the 75th amino acid residue(Leu) of the sequence of formula (I), a gene coding for the amino acidsequence from the 1st to the 77th amino acid residue (Asn) of thesequence of formula (I), a gene coding for the amino acid sequence fromthe 1st to the 82nd amino acid residue (Leu) of the sequence of formula(I) and a gene coding for the amino acid sequence from the 1st to the86th amino acid residue (Thr) of the sequence of formula (I). Inaddition, mention may be made also of a gene coding for the amino acidsequence from the 1st to the 103rd amino acid residue (Pro) of thesequence of formula (I). When higher animal cells are used as host cellsfor its expression, this gene gives human MACIF with the PI anchorattached to the 76th amino acid residue (Glu), when expressed inbacteria, for instance, the gene gives a modified human MACIF proteincomprising a peptide up to the 103rd amino acid residue (Pro); the PIanchor attachment does not occur in that case. Therefore, forconvenience sake, such gene is included in the category of modifiedprotein-encoding genes. Furthermore, a gene coding for the amino acidsequence up to the 76th amino acid residue (Glu) which constitutes thepeptide core of human MACIF, when expressed on a vector, gives a solublehuman MACIF polypeptide portion having no PI anchor. Such gene may alsobe included in the category of modified protein-encoding genes asdefined above. In addition to those genes specifically mentioned above,genes coding for the amino acid sequence from the 1st up to the 71st to85th amino acid residue can produce proteins having human MACIF activityand therefore are included among the genes according to the invention.

The genes according to the invention can be prepared by various methods.For example, a suitable method comprises isolating a clone containingcDNA coding for human MACIF from a cDNA library prepared from mRNAobtained from human MACIF-producing cells and isolating the MACIF cDNAfrom the thus-isolated clone. Another method comprises chemicallysynthesizing nucleic acids in the conventional manner, for example bythe phosphoamidite method [Hunkapiller, M. et al., Nature, 310, 105-111(1984)], based on the genetic information encoding human MACIF asdisclosed herein. A combination of the above two methods may bementioned as a further example. In the following, the method mentionedabove that makes use of mRNA is described in further detail.

Oligonucleotide probe preparation

An oligodeoxyribonucleotide probe is prepared which is complementary tomRNA coding for the N-terminal amino acid sequence of purified,naturally occurring human MACIF isolated from human erythrocytes, forexample, by chemical synthesis by the phosphoamidite method using acommercial DNA synthesizer (e.g., Applied Biosystems model 380A DNAsynthesizer).

cDNA library preparation

(1) Preparation of raw material cells:

Human cells of any kind in which the human MACIF according to theinvention can be expressed may be used as the material for human MACIFmRNA in the practice of the invention. Advantageous as such cells fromthe high mRNA content viewpoint are human peripheral blood-derivedleukocytic cells, human lymphocytic cells and other tissue cells as wellas appropriate cell lines established therefrom.

Human peripheral blood-derived leukocytes and lymphocytes can beisolated from a normal human-derived buffy coat by density gradientcentrifugation [Boyum, A., Scandinavian Journal of Clinical LaboratoryInvestigations, 21, Supplement 97, 77-89 (1968)] using dextran orFicoll-Hypaque, for instance. Tissue cells can be prepared from a tissuehomogenate or the like in the conventional manner.

As the established human cell lines, there may be mentioned, forexample, human erythroblastic leukemia cell lines (e.g., K562), human Bcell leukemia cell lines (e.g., Raji), human T cell leukemia cell lines(e.g., MT-2), human monocytic leukemia cell lines (e.g., U937, HL60) andother tissue cancer cell lines (e.g., Bowes melanoma). The cell lines tobe used are not limited to these, however. Such cell lines are readilyavailable from the Salk Institute Cell Bank (California, U.S.A.) andother similar institutions. The cells are cultured in the manner ofstationary culture, spinner culture or roller bottle culture, forinstance, using an appropriate animal cell culture medium, e.g.,commercially available RPMI 1640 medium [Moore, G. E. et al., Journal ofAmerican Medical Association, 199, 519-524 (1967)].

In some instances, stimulation of cells during cultivation can result inintracellular expression of human MACIF mRNA in increased amounts. Theuse of an immune complex as the stimulant is advantageous. As thestimulant, there may be further mentioned lectins [e.g., concanavalin A(ConA), phytohemagglutinin (PHA)], various antigens, phorbol esters [inparticular 12-O-tetradecanoylphorbol-13-acetate (TPA)] and physiologicalstimulatory factors (e.g., interleukins, interferons, colony stimulatingfactors), among others. These may be used in combination of two or threeof them.

(2) mRNA extraction:

An RNA fraction containing human MACIF-encoding mRNA can be extractedfrom cells of any of the kinds mentioned above in the conventionalmanner. Thus, for instance, the cells are partially or completelydisrupted and/or solubilized by means of a guanidine thiocyanatesolution or an appropriate detergent (e.g., SDS, NP-40, Triton X-100,deoxycholic acid) or by a physical means such as homogenization orhemolysis. Chromosomal DNA is then subjected, to a certain extent, tothe shearing action of a mixer (e.g., Polytron) or a syringe, followedby separation of a nucleic acid fraction from proteins. Particularlyuseful for this fractionation procedure is the technique of extractionwith phenol and chloroform or cesium chloride density gradientultracentrifugation [Chirgwin, J. M. et al., Biochemisty, 18, 5294-5299(1979); Maniatis, T. et al., Molecular Cloning, A Laboratory Manual,Cold Spring Harbor Laboratory, (1982)], among others.

In the above-mentioned extraction procedure, an RNAase inhibitor, forexample, heparin, polyvinyl sulfate, diethyl pyrocarbonate or a vanadiumcomplex, may be used as an additive for preventing RNA degradation dueto RNase.

Isolation and purification of mRNA from the RNA obtained by the aboveextraction procedure can be effected, for example, by using anadsorption column of, for example, oligo-dT-cellulose (CollaborativeResearch) or poly-U-Sepharose (Pharmacia), or in a batchwise manner.

The thus-obtained mRNA is a mixture of mRNAs coding for a variety ofproteins. Therefore, it may be purified and concentrated with respect tothe desired mRNA that corresponds to human MACIF prior to cDNA librarypreparation. This purification and concentration can be performed asfollows. Thus, the mRNA obtained in the above manner is fractionated bysucrose density gradient centrifugation, for instance, and the resultingfractions are tested for the presence of the desired human MACIF mRNA bydot plot hybridization, for instance.

(3) cDNA Library preparation:

The purified mRNA obtained in the above manner is generally unstable.Therefore, the mRNA is converted (reverse transcribed) to a stablecomplementary DNA (cDNA) and connected to a microorganism-derivedreplicon, enabling amplification of the desired gene. The in vitro mRNAconversion can be generally carried out by the Okayama-Berg method[Okayama, H. and Berg. P., Molecular and Cellular Biology, 2, 161-170(1982)].

Thus, oligo-dT is used as a primer, which may be free oligo-dT or in theform already attached to a vector primer, and a single-stranded cDNAcomplementary to the mRNA is synthesized using the mRNA as a templateand using reverse transcriptase in the presence of dNTPs (dATP, dGTP,dCTP and dTTP). The next step depends on whether the oligo dT is used inthe free form or in the form attached to a vector primer.

In the former case, the template mRNA is removed by decomposing it bytreatment with an alkali, for instance, and then a double-stranded DNAis synthesized in the presence of reverse transcriptase or DNApolymerase I with the single-stranded DNA as a template. The resultantdouble-stranded DNA is then treated at both ends thereof with SInuclease and, after addition of an appropriate linker DNA or a pluralityof bases whose combination allows annealing to the respective ends,inserted into an appropriate vector, for example, an EK system plasmidvector (either of the stringent type or of the relaxed type), or a λgtphage vector.

In the latter case, the mRNA to serve as a template is allowed to remainas it is, and the opened circular plasmid with the same linkers asmentioned above added thereto is annealed with a linker DNA (frequentlya DNA fragment containing a region autonomously replicable in cells andan mRNA transcription promoter region) to give a closed circular form.Then, the mRNA is replaced with a DNA chain in the presence of dNTPs andin the simultaneous presence of RNase H and DNA polymerase I to give acomplete plasmid DNA.

The cDNA-containing plasmid vector obtained in the above manner can beintroduced into a host for transformation thereof. Typical as the hostis Escherichia coli. However, the host is not limited to this but maybe, for example, Bacillus subtilis or Saccharomyces cerevisiae.

The host can be transformed by introducing the DNA mentioned above byvarious methods commonly used in the art, for example, by collectingcells mainly at the logarithmic growth phase, treating them with calciumchloride for rendering them ready for spontaneous DNA uptake andallowing them to take up the plasmid. In the procedure mentioned above,magnesium chloride or rubidium chloride may be allowed to additionallycoexist in the transformation system for further improvement of thetransformation efficiency, as is generally known in the art. It is alsopossible to convert host cells to the spheroplast or protoplast stateprior to transformation.

Cloning of cDNA

A strain carrying the desired human MACIF cDNA can be detected fromamong the transformants obtained in the above manner by various methods,for example, the methods mentioned below.

(1) Screening using a synthetic oligonucleotide probe:

In cases where a part of the amino acid sequence of the desired proteinis known, as in the case of the present invention, oligonucleotidescorresponding to the amino acid sequence portion is synthesized and thisis used as a probe (after labeling with ³² P or ³⁵ S) for detecting andselecting a positive strain by hybridization with transformant-derivedDNAs denatured and immobilized on a nitrocellulose filter. Theoligonucleotide may have a base sequence derived based on the codonfrequency data or a combination of presumable base sequences. In thelatter case, the number of probes can be reduced by incorporatinginosine thereinto.

(2) Selection using an antibody to human MACIF:

The cDNA is inserted in advance into a vector capable of proteinexpression in transformants, protein production is allowed to proceedtherein, and the anti-human MACIF antibody and a second antibody to theantibody are used to detect and select a desired human MACIF producercell.

(3) Screening by producing human MACIF in other animal cells:

Transformant strains are cultured for gene amplification. The genes areused to transfect animal cells (using a plasmid capable of autonomouslyreplicating and containing an RNA transcription promoter region or aplasmid capable of integrating into animal cell chromosomes). Theproteins encoded by the genes are allowed to be produced and the culturesupernatants or cell extracts are assayed for human MACIF activity.Alternatively, a transformant strain carrying the desired cDNA codingfor human MACIF is selected from among transformant strains by detectingthe human MACIF produced therein by using an antibody to human MACIF.

Confirmation of human MACIF cDNA

The gene according to the present invention obtained by the use of mRNAas a starting material can be confirmed to be a gene properly coding forhuman MACIF by using an appropriate translation system. Most commonlyused is the method developed by Krieg et al. [Krieg, P. A. et al.,Nucleic Acids Research, 12, 7057-7070 (1984)], which comprisessynthesizing a large quantity of mRNA in vitro using a potent promoterand an RNA polymerase specific to the promoter, followed by translationof the mRNA into a protein using a simple translation system. Thus, inthe above method, the cDNA of the present invention is inserted into anappropriate plasmid downstream from a potent promoter, such as the SP6promoter, T7 promoter or T3 promoter, (in the case of the use of mRNA asa starting material, the vector containing these promoters can bepreviously used in the library preparation), and the resulting plasmidis purified and then cleaved at an appropriate restriction enzymecleavage site occurring downstream from the human MACIF cDNA whichoccurs downstream from such promoter. The resulting double-stranded DNAis transcribed to mRNA in vitro using a polymerase specific to thepromoter used, such as SP6 polymerase, T7 polymerase or T3 polymerase,respectively. The thus-transcribed mRNA is then translated into aprotein by using a cell-free protein synthesizing system, such as arabbit reticulocyte lysate or wheat germ lysate, or by the methodcomprising injecting the mRNA into Xenopus laevis oocytes. That the genecorrectly coding for human MACIF has been obtained can be confirmed byassaying for MACIF activity of the translation product protein, or by animmunological method using an antibody specific to human MACIF.

Gene sequence determination

The nucleotide sequence of the thus-obtained gene of the presentinvention can be determined, for example, by the dideoxy method using aplasmid vector [Chen, E. Y., DNA, R, 165-170 (1985)] or by the 7-DEAZAmethod [Mizusawa, S. et al., Nucleic Acids Research, 14, 1319-1324(1986)]. The thus-obtained gene for naturally occurring human MACIF(derived from human leukocytes) is shown in FIG. 1.

While the method of preparing the gene of the present invention asdetailedly described above goes via mRNA, the gene coding for humanMACIF can also be prepared by chemical synthesis based on the nucleotidesequence disclosed herein. A typical example of the method of chemicalsynthesis is the phosphoamidite method.

Construction of expression vectors

(1) Selection of the host-vector system:

The whole length of the coding region of the thus-obtained human MACIFgene can be expressed using a eukaryote or prokaryote as the host. Thevector to be integrated into such host cells can be constructed in anappropriate manner depending on the host cells.

As the prokaryote host, there may be mentioned Escherichia coli strains[e.g., E. coli K12 294 (ATCC 31446), E. coli B, E. coli X1776 (ATCC31537), E. coli C600, E. coli W3110 (F-, λ-, prototrophic; ATCC 27375)],Bacillus strains [e.g., B. subtilis], enteric bacteria other than E.coli, for example, Salmonella typhimurium and Serratia marcescens, andPseudomonas strains.

Usable as the vector for such microorganism hosts is an expressionvector which contains the gene of the present invention with a promoterand an SD base sequence [Shine, J. et al., Proc. Natl. Acad. Sci.U.S.A., 71, 1342-1346 (1974)] located upstream of the gene, togetherwith ATG necessary for protein synthesis initiation. Generally, pBR322,pBR327 and the like are vectors suited for use in Escherichia coli andother microbial strains.

Usable as the promoter are, for example, the tryptophan promoter, PLpromoter, lac promoter, 1pp promoter and β-lactamase promoter.

As typical examples of the marker gene, there may be mentioned theampicillin resistance gene and tetracycline resistance gene.

Yeasts are generally used as the eukaryotic microorganism. Inparticular, yeasts belonging to the genus Saccharomyces can be usedadvantageously. A typical example of the expression vector for use inyeasts and other eukaryotic microorganisms is YRp7.

Useful examples of the promoter which the expression vector forexpression in yeasts should have are the 3-phosphoglycerate kinase,enolase, glyceraldehyde-3-phosphate dehydrogenase or hexokinasepromoters.

The trp1 gene, for example, can be used as the marker gene.

The origin of replication, termination codon and other DNA sequenceswhich serve to regulate transcription and translation in yeast cells maybe ordinary DNA sequences known to be suited for use in yeast cells.

When cultured higher animal cells are used as the host, they may berhesus monkey kidney cells, mosquito larva cells, African green monkeykidney cells, mouse fetus fibroblasts, Chinese hamster ovary (CHO)cells, a dihydrofolate reductase-deficient strain thereof [Urlaub, G. etal., Proc. Natl. Acad. Sci., U.S.A., 77, 4216-4220 (1980)], humancervical epithelial cells, human fetus kidney cells, moth ovary cells,human myeloma cells or mouse fibroblasts, for instance.

The vector generally contains functional sequences for expression of theDNA of the present invention in host cells, for example, the origin ofreplication, promoter located upstream from the DNA of the presentinvention, ribosome binding site, polyadenylation site and transcriptiontermination sequence.

Preferred examples of the promoter are the adenovirus 2 major latepromoter, SV40 early promoter, SV40 late promoter, eukaryoticgene-derived promoters (e.g., estrogen-inducible chicken egg albumingene, interferon gene, glucocorticoid-inducible tyrosineaminotransferase gene, thymidine kinase gene, adenovirus major early andlate genes, phosphoglycerate kinase gene, α-factor gene).

The origin of replication may be derived from adenovirus, SV40, bovinepapilloma virus (BPV), vesicular stomatitis virus (VSV), or any ofvectors derived from these.

The neomycin resistance gene, and methotrexate resistant dihydrofolatereductase (DHFR) gene, among others, can be used as the marker gene inthis case.

The examples of the host, vector and constituent elements thereof thathave been described hereinabove as usable for the expression of thehuman MACIF cDNA and modified human MACIF protein cDNAs are by no meanslimitative of the scope of the present invention.

(2) Construction of human MACIF expression vectors:

Since, as mentioned hereinbefore, human MACIF is a protein having the PIanchor at the C terminus of the polypeptide chain, the host cells forits expression must be selected from among cells having the PI anchorsynthesizing mechanism. Such mechanism is known to be distributed amonga wide variety of organisms from prokaryotes, yeasts and myxomycetes toinsects and mammals. As examples of the host cells that can be expectedto allow production of a polypeptide having the PI anchor at the Cterminus thereof in the state of art, there may be mentioned CHO cells[Caras, I. W. et al., Nature, 322, 545-549 (1987)], COS cells [Caras, I.W. et al., Science, 243, 1196-1198 (1989)] and R1.1 thymoma cells[Waneck, G. L. et al., Science, 241, 697-699 (1988)], but the host cellsare not limited thereto. In the following, a method of constructing ahuman MACIF cDNA expression vector particularly suited for theexpression in Chinese hamster ovary cells (CHO cells) as host cells isdescribed in detail.

A human MACIF cDNA-containing clone, PGEM352-3, is isolated from humanmonocyte cDNA library constructed with commercially available plasmidvector PGEM4 (Promega) mainly used for in vitro transcription. Theplasmid pGEM352-3, is cleaved with the restriction enzymes SacI andHincII, followed by agarose gel electrophoresis to give a SacI/HincIIDNA fragment of about 425 base pairs. This fragment is renderedblunt-ended by treatment with mung bean nuclease.

On the other hand, the main body vector for constructing an expressionvector for use in CHO cells is prepared by cleaving pVY-1 (shown in FIG.4) with BglII, followed by treatment with mung bean nuclease to renderthe resulting BglII ends blunt-ended. This main body vector and theblunt-ended SacI/HincII DNA fragment mentioned above are joined togetherusing T4 DNA ligase, and the ligation mixture is used to transformEscherichia coli HB101. Plasmids are prepared from the thus-obtainedtransformants by the alkali method and subjected to restriction enzymeanalysis. In this way, a plasmid capable of expressing the gene isselected out.

The thus-prepared expression plasmid is transformed into methotrexate(Mtx)-susceptible CHO cells by the calcium phosphate method. Sincetransformants acquire Mtx resistance, a strain capable of expressing thepolypeptide can be selected from among the Mtx-resistant strains. Whenthe gene is expressed in CHO cells, the expression product polypeptideis deprived of the signal peptide, with or without further processing,to give mainly a protein having the PI anchor attached to the Cterminus.

A vector for the expression of human MACIF having thephosphatidylinositol anchor at the C terminus can be constructed also bythe known technology mentioned hereinbelow using a modification of theabove gene modified in the portion which codes for the hydrophobicsignal sequence for PI anchor attachment beginning with the 77th aminoacid residue (Asn) of the polypeptide of formula (I).

From the teaching of Caras et al. [Caras, I. W. et al., Science, 238,1280-1283 (1987)], it is well known in the art that thephosphatidylinositol anchor can be attached to the C terminus of anydesired protein when a hybrid DNA is constructed by adding a DNA codingfor the hydrophobic signal sequence for PI anchor attachment at the Cterminus of a precursor for a known PI-anchored protein to the 3'terminus of a DNA coding for a desired protein and then expressed usinga recombinant vector such as mentioned hereinbefore. The hydrophobicsignal sequence for PI anchor attachment may be any of those known PIanchor attachment signal sequences for precursors for PI-anchoredproteins that are described in a review by Ferguson et al. [Ferguson, M.A. et al., Annual Review of Biochemistry, 57, 285-320 (1988)] andelsewhere.

According to Caras et al. [Caras, I. W. et al., Science, 243, 1196-1198(1989)], it is possible that the N-terminal secretory signal sequence ofhuman growth hormone or a random hydrophobic peptide sequence can beused as a polypeptide sequence usable as a signal for PI anchorattachment at the C terminus. This suggests that human MACIF with the PIanchor attached thereto can be expressed as well by using a hybrid geneproduced by connecting such a hydrophobic sequence as mentioned above tothe 3' side of a DNA sequence coding for the portion of human MACIFwhich ends in the 76th amino acid residue (Glu).

In view of the above, it is evident that the gene sequence to be used inconstructing an expression vector for the production of human MACIF withthe PI anchor attached to the C terminus thereof is not limited to theDNA sequence shown in FIG. 1.

The human MACIF polypeptide with the PI anchor attached to the Cterminus thereof is generally expressed on the cell membrane of thetransformant. Therefore, the recombinant human MACIF on the cell surfacecan be detected in the conventional manner by reacting the transformantcells with an anti-human MACIF antibody and a fluorescence-labeledsecond antibody, followed by flow cytometry or analysis on afluorescence-activated cell sorter (FACS). If no human MACIF can bedetected on the cell surface any more by the above method aftertreatment of the transformant cells with phosphatidylinositol-specificphospholipase C (PI-PLC), it can conversely be verified that therecombinant MACIF obtained in the above manner has the PI anchorattached thereto.

Such a method as mentioned above makes it possible to confirm that arecombinant human MACIF is produced in cells transformed in the abovemanner.

(3) Construction of expression vectors for modified human MACIF protein:

A method of constructing vectors for the expression of modified humanMACIF proteins is described in the following.

These are recombinant vectors capable of expressing proteinssubstantially equivalent in physiological activities to human MACIF.They are vectors comprising a DNA sequence derived from the DNA basesequence shown in FIG. 1 by deleting any portion thereof, for example,such that the expression product lacks in a part of its N-terminal orC-terminal amino acid sequence, or by modifying any portion thereof suchthat the corresponding amino acid sequence be replaced by some otheramino acid sequence, or by adding an appropriate sequence such that someamino acid sequence be added to the expression product, together withthose constituent elements necessary for expression in a variety of hostsystems mentioned in the above section (1). More specifically, theseexpression vectors include a series of recombinant vectors in which agene coding for a polypeptide defined by the amino acid sequence offormula (I) is effectively connected to and placed under the control ofa regulatory DNA sequences capable of causing expression of thepolypeptide.

When bacterial cells, which have no PI anchor synthesizing mechanism,are used as the host for expression of the above-mentioned expressionvectors, the expression product is always a polypeptide having no PIanchor.

When mammalian cells are used as the host, the PI anchor addition iscontrolled in a complicated manner. It is generally known that when PIanchor attachment to the C terminus of a polypeptide takes place,elimination of a specific hydrophobic C-terminal polypeptide portion(PI-anchor attachment signal sequence) from a precursor for thepolypeptide precedes modification with the PI anchor. In the case ofhuman MACIF, the polypeptide from the 77th amino acid residue (Asn) tothe 103rd amino acid residue (Pro) acts as a signal for PI anchorattachment. However, the respective modified protein genes inserted inthe expression vectors for those modified proteins mentioned above whichare composed of amino acid residues No. 1 to No. 70 to 86 are lacking inthe portion of DNA which codes for a part or the whole of the PI anchorattachment signal sequence. It is known that deletion of a gene fragmentcoding for a part of the hydrophobic attachment signal sequence from agene coding for a protein precursor to which a PI anchor is to be addedresults in production of a soluble protein [Berger, J. et al., Journalof Biological Chemistry, 263, 10016-10021 (1988)]. Therefore, thesegenes of the present invention which code for the modified proteins canpossibly be used as genes for producing soluble modified human MACIFproteins.

Among the vectors for the expression of these modified proteins, avector for the expression in Escherichia coli of a protein comprisingthose amino acid residues up to the 103rd one shown in FIG. 2, vectorsfor the expression in Escherichia coli and in mammalian cells ofmodified MACIF proteins comprising those amino acid residues up to the86th, 77th and 70th, respectively, are described in more detail in thefollowing together with methods of their construction and expression.

(a) Expression in Escherichia coli of a protein comprising the 1st to103rd amino acid residues:

The plasmid pGEM352-3 is cleaved with the restriction enzymes PstI andHincII and a PstI/HincII DNA fragment of about 310 base pairs isisolated and purified by agarose gel electrophoresis.

A synthetic DNA of the formula (II): ##STR4## is phosphorylated at thePstI cleavage site is joined to the PstI/HincII fragment prepared asabove-mentioned at the PstI cleavage site thereof using T4 DNA ligase.The thus-produced, synthetic DNA-joined DNA fragment (now EcoRI/HincIIfragment) is isolated and purified by agarose gel electrophoresis.

Separately, a vector main body for constructing an expression vector foruse in Escherichia coli is prepared, for example, by cleaving pKK223-3shown in FIG. 3 with EcoRI and SmaI. This vector and the above-mentionedEcoRI/HincII DNA fragment are ligated together using T4 DNA ligase andthe ligation mixture is used to transform E. coli K12JM109 by thecalcium chloride method. Plasmids are isolated from transformants by thealkaline method and analyzed using restriction enzymes. In this way, atransformant harboring the desired plasmid with the EcoRI/HincII DNAfragment inserted therein can be selected.

The desired polypeptide can be expressed and produced by cultivating thetransformant obtained as described above in an appropriate medium (e.g.,L medium containing 100 mM isopropyl-β-D-thiogalactopyranoside). Thepolypeptide expressed has methionine residue at the N terminus thereof.The expression product also includes the polypeptide species which hasno N-terminal methionine as a result of elimination by the action of anenzyme occurring in Escherichia coli cells and capable of eliminatingthe N-terminal methionine residue.

(b) Expression of a protein comprising the 1st to 86th amino acidresidues:

When Escherichia coli is used as the host, pGEM352-3 is cleaved withAvaII. Separately, a synthetic DNA of the formula (III): ##STR5## isphosphorylatd at the AvaII cleavage site. The cleavage product andphosphorylated synthetic DNA are ligated together using T4 DNA ligase.The ligation product is then cleaved with PstI and ligated with thesynthetic DNA of formula (II) phosphorylated at the PstI cleavage site,using T4 DNA ligase. The thus-produced ligation product (now EcoRI/HindIII fragment) from the synthetic DNA and DNA fragment is isolated andpurified by agarose gel electrophoresis.

The thus-prepared EcoRI/HindIII DNA fragment is ligated with anEcoRI/HindIII fragment of pKK223-3. A recombinant Escherichia colistrain, which allows expression of a polypeptide comprising the 1st to86th amino acid residues, can be obtained using the ligation product andproceeding in the same manner as described above.

For polypeptide expression in CHO cells, pGEM352-3 is cleaved with AvaIIand then ligated with the synthetic DNA of formula (III), as in the caseof expression in Escherichia coli cells. The ligation product is thencleaved with SacI and a DNA fragment of about 375 base pairs is isolatedand purified by agarose gel electrophoresis. This DNA fragment isrendered blunt-ended by treatment with mung bean nuclease. The resultingDNA fragment is inserted into pVY1 cleaved with BglII and renderedblunt-ended to give an expression plasmid. Transformation of CHO cellswith the expression plasmid gives CHO cells capable of expressing andproducing a polypeptide comprising the 1st to the 86th amino acidresidues.

(c) Expression of proteins comprising the 1st to 77th amino acidresidues and the 1st to 70th amino acid residues, respectively:

For causing expression of the gene coding for the 1st to 77th amino acidresidues or the 1st to 70th amino acid residues in Escherichia coli orCHO cells, essentially the same procedure as mentioned above can befollowed except that, in the plasmid preparation step, the synthetic DNAto be joined to pGEM352-3 after cleavage of the latter with AvaII is asynthetic DNA of the formula (IV): ##STR6## when the polypeptidecomprising the 1st to the 77th amino acid residues is to be expressed,or a synthetic DNA of the formula (V): ##STR7## when the polypeptidecomprising the 1st to the 70th amino acid residues is to be expressed.

The subsequent steps, the corresponding genes and recombinant vectorsfor the expression of the genes can be obtained by proceeding in thesame manner as described above in (b).

Transformation

Introduction of the thus-obtained expression vectors containing thehuman MACIF cDNA or modified human MACIF protein cDNA into desired hostcells, namely transformation of the cells with the vectors, can beeffected using these techniques that are used generally.

Each expression vector plasmid can be prepared from the host used forgene construction (e.g. E. coli HB101) by a method in general use, forexample, alkaline bacteriolysis. The vector plasmid prepared is used totransform the host. The transformation can be effected by the method ofHanahan [Hanahan, D., Journal of Molecular Biology, 166, 557-580(1983)], for instance, when bacterial cells are used as the host, or bythe calcium phosphate method [van der Eb, A. J. et al., Methods inEnzymology, 65, 826-839 (1980), Academic Press], for instance, whenmammalian cells are used as the host.

Cultivation and purification

The transformant obtained in the above manner can be grown in theconventional manner and cultivation thereof results in production andaccumulation of a biologically active human MACIF or modified humanMACIF proteins. The medium for the cultivation can suitably be selectedfrom among various conventional media depending on the host cellsemployed. For instance, when the above-mentioned CHO cells are used asthe host, MEM-α medium if necessary supplemented with a blood componentsuch as fetal calf serum (FCS), may be used.

The site of expression for the production of the recombinant human MACIFor recombinant modified human MACIF proteins in the transformant differsdepending on the amino acid sequence encoded by the cDNA selected, thekind of vectors, that of the host cells and the combination of these.Thus, the recombinant human MACIF or modifications thereof can beproduced on the cell membrane, within the cell or in the cell culturesupernatant. The human MACIF or modified human MACIF proteins producedin transformed cells can be isolated and purified therefrom by variousseparation techniques [e.g., Japanese Biochemical Society (ed.),Biochemical Data Book II, 1st edition, 1st printing, page 1175, TokyoKagaku Dojin (1980)] based on the physical and chemical propertiesthereof.

To be concrete, the techniques include, among others, treatment with anordinary protein-precipitating agent, ultrafiltration, molecular sievechromatography (gel filtration), adsorption chromatography, ion exchangechromatography, affinity chromatography, high-performance liquidchromatography (HPLC), other liquid chromatographic techniques,dialysis, and combinations of these. To be more concrete, the techniqueto be used may vary depending on the site of expression of therecombinant protein.

Recombinant proteins produced in the culture supernatant can be isolatedand purified in the following manner.

First, the desired substance is partially purified from the culturesupernatant in advance. This partial purification is realized, forexample, by treatment with an agent for salting out, such as ammoniumsulfate, sodium sulfate or sodium phosphate, and/or ultrafiltrationtreatment using a dialyzing membrane, flat membrane or hollow fibermembrane. The procedure and conditions of each of these treatments maybe the same as those generally employed in the art. The roughly purifiedproduct obtained in the above manner is subjected to adsorptionchromatography, affinity chromatography, gel filtration, ion exchangechromatography, reversed-phase chromatography or the like or acombination of these, and a fraction showing human MACIF activity isrecovered. In this manner, the desired substance can be isolated in apure and homogeneous form.

A recombinant protein produced on the cell membrane may have a PI anchorand, therefore, may be bound to the cell membrane via the anchor. Suchmembrane-bound recombinant protein can be purified by disrupting thecell membrane by treatment with an appropriate detergent (e.g. NP-40,Triton X-100, octylglycoside) and then proceeding in the same manner asdescribed above. Alternatively, a recombinant protein bound to the cellmembrane via the PI anchor can be solubilized by an appropriatetreatment for PI anchor cleavage. As the means of PI anchor cleavage,there may be mentioned, for example, cleavage withphosphatidylinositol-specific phospholipase C (PI-PLC) and cleavage withphosphatidylinositol-specific phospholipase D (PI-PLD). The recombinantprotein solubilized by the above treatment is released into the cellculture supernatant and therefore can be purified in the same manner asdescribed above.

A recombinant protein produced in the cell can be purified by disruptingthe cell membrane by treatment with an appropriate detergent, as in thecase of membrane-bound recombinant proteins, to thereby cause release ofthe recombinant protein into the solution phase and then proceeding inthe same manner as mentioned above.

The activity of the thus-purified recombinant human MACIF, solublerecombinant human MACIF or recombinant modified human MACIF proteins canbe identified, for example, by measuring of reactive lysis [Thompson, R.A. et al., Journal of Experimental Medicine, 131, 629-641 (1970) andLachmann, P. J. et al., Journal of Experimental Medicine, 131, 643-657(1970)] inhibiting activity assay.

In the foregoing, the genes according to the present invention, thevectors for the expression of the genes and the transformedmicroorganisms and cells capable of allowing the expression of the genesas well as the methods for their preparation have been described. As aresult of the present invention, the DNA sequence of the human MACIFgene has been determined for the first time and at the same time genescoding for modified human MACIF proteins practically useful as drugshave been provided. Thus, it is now possible to produce a pure andhomogeneous grade of human MACIF or a modification thereof in largequantities by using the recombinant DNA technology.

Human MACIF and modified human MACIF proteins are of the human origin.Therefore, they have no antigenicity and their toxicity is low. They canprevent cells and tissues from being damaged as a result of MACformation in the last stage of complement activation. Thus, they can beused advantageously as therapeutic agents for various diseases, inparticular diseases resulting from the absence or reduced level of acomplement-regulating component(s) and all diseases classifiable underthe category of type II or type III allergy.

Furthermore, not only in disease due to the absence or reduced level ofone or more complement-regulating components, typically in paroxysmalnocturnal hemoglobinuria (PNH), but also in various inflammatory orautoimmune diseases accompanied by cell and/or tissue damage, aqualitative or quantitative abnormality in human MACIF may possibly beobserved. Therefore, human MACIF and modified proteins derivedtherefrom, monoclonal or polyclonal antibodies specific to human MACIFor a modification thereof, human MACIF or modified human MACIF proteinDNAs and, further, DNAs complementary to the human MACIF or modifiedhuman MACIF protein genes can be used in specific diagnosis of theabove-mentioned diseases.

The dosage form of human MACIF or modified human MACIF proteins may varydepending on the diseases, symptoms and patient's conditions. Generally,however, non-oral dosage forms, such as injections, nasal preparations,suppositories and implants, are used for systemic administration, whileintraarticular preparations and preparations to be implanted intoaffected sites, for instance, are used for local administration.

In preparing these dosage forms, compositions suited for the respectiveforms are used. In preparing injections, human MACIF or a modified humanMACIF protein is dissolved in phosphate-buffered physiological saline oran aqueous dextrose solution, for instance, and, after addition of astabilizer, a dispersant and/or the like, as necessary, distributed intoampules or lyophilized in vials. In the latter case, the preparation isreconstituted prior to use by dissolving in distilled water forinjection or physiological saline.

The daily dose of MACIF for human adults is generally within the rangeof 100 μg to 5,000 mg, preferably within the range of 1 mg to 500 mg.Such daily dose is administered in a single dose or in divided doses.

Dosage form preparation example

A solution of 5 g of MACIF in 100 ml of physiological saline issubjected to aseptic filtration and then distributed in 2-ml portionsinto vials. Lyophilization gives a preparation for injection, each vialcontaining 100 mg of MACIF.

EXAMPLES

(1) Determination of N-terminal and C-terminal amino acid sequences of apurified sample of naturally occurring human MACIF:

1) N-terminal amino acid sequence determination

A purified sample of naturally occurring human MACIF (18 kilodaltons)was reduced in the conventional manner with 2-mercaptoethanol in thepresence of 8M urea, then S-carboxymethylated with iodoacetic acid, andanalyzed for its N-terminal amino acid sequence using a gas phaseprotein sequencer (model 470A, Applied Biosystems, U.S.A.). The resultwas as follows: ##STR8## 2) C-terminal amino acid sequencedetermination:

A 0.6 mg portion of the purified sample of naturally occurring humanMACIF was reduced in the conventional manner with dithiothreitol in thepresence of 6M guanidine hydrochloride and S-carboxymethylated withiodoacetic acid. The alkylation mixture was dialyzed against distilledwater overnight at 4° C. and the dialyzate was concentrated to 0.5 mlusing a centrifuge-type reduced-pressure concentrator. The wholecarboxymethylated human MACIF solution was buffered by addition of 1MTris hydrochloride buffer (pH 8.0). Then, a solution of 100 μg ofPronase (Calbiochem) in 20 μl of 50 mM Tris hydrochloride buffer wasadded to the solution, and the reaction was allowed to proceed at 37° C.for 22 hours.

A chloroform-methanol (1:1) mixture (0.5 ml) was added to the reactionmixture. After thorough shaking, the whole mixture was centrifuged,whereupon it separated into a transparent upper layer, a cloudy middlelayer and a transparent lower layer. A portion of each layer was taken,hydrolyzed with 6N hydrochloric acid and analyzed in the conventionalmanner using a Picotag work station equipment (Waters, U.S.A.).Ethanolamine was detected in the middle layer. It was thus revealed thatthe middle layer contained a Pronase digestion fragment with a PI anchorattached at the C terminus. The middle layer (600 μl) containing theabove PI anchor-attached C-terminal fragment was lyophilized, 1.2 ml ofa water:n-butanol:1N hydrochloric acid (600:600:3) mixture was addedand, after thorough shaking, the mixture was centrifuged. Hydrolysis wasperformed with 6N hydrochloric acid in the same manner as describedabove and analyzed using a Picotag work station equipment. Ethanolaminewas detected in the butanol layer (upper layer). An equal volume ofn-butanol-saturated 5 mM hydrochloric acid was added to the upper layer,the mixture was shaken and then centrifuged and the upper layer wasseparated. This extraction procedure with acidic butanol was repeatedtwice in all.

Half of the final n-butanol layer was subjected to amino acid sequencedetermination using the protein sequencer mentioned above.

As a result, the following sequence was found: ##STR9## this covers fromthe 72nd to the 76th amino acid residue in the sequence shown in FIG. 2.No amino acid residue was detected behind the 76th amino acid residue(Glu). The above facts indicated that erythrocyte-derived, naturallyoccurring human MACIF has the PI anchor attached to the Glu which is the76th amino acid residue from the N terminus.

(2) Preparation of oligonucleotide probes for detecting cDNA clonescoding for human MACIF:

Based on the amino acid sequence revealed in the above section (1),15-mer and 17-mer mixed deoxyoligo-nucleotide probes complementary tothe mRNA regions coding for amino acid residues Nos. +1 to +5 and +4 to+9, respectively, were chemically synthesized by the phosphoamiditemethod using a model 380A DNA synthesizer (Applied Biosystems) andlabeled with ³² P at the 5' end.

The 15-mer mixed probe (hereinafter referred to as "M1 probe") had thenucleotide sequences: ##STR10## while the 17-mer mixed probe(hereinafter referred to as "M4 probe") had the nucleotide sequences##STR11## (3) Preparation of a human monocyte cDNA library:

Recombinant plasmids were constructed by the Okayama-Berg method (videsupra) starting with mRNA derived from immune complex-stimulated humanperipheral blood monocytes and the commercially available plasmid vectorpGEM4 (Promega). The recombinant plasmids thus constructed had cDNAinserted between the KpnI cleavage site and SacI cleavage site of themulticloning sites occurring between the SP6 promoter region and T7promoter region of pGEM4. The directionality of cDNA was such that theKpnI side (T7 promoter side) was on the 3' side of mRNA and the SacIside (SP6 promoter side) was on the 5' side of mRNA.

The recombinant plasmid mixture thus obtained was used to transform E.coli HB101 competent cells (Takara Shuzo) and a cDNA library comprisingabout 400,000 transformants was obtained.

(4) Cloning of human MACIF cDNA:

For isolating a transformant harboring a plasmid containing cDNA codingfor human MACIF, the human monocyte cDNA library obtained as describedin section (3) was subjected to colony hybridization using the syntheticoligonucleotide probes prepared as described in section (2) to detectclones hybridizing with both of M1 and M4 probes. A clone containing thelongest cDNA (about 2,000 bp) was selected and, for the plasmid pGEM352-3 isolated therefrom, a partial base sequence of the cDNA portionwas determined by the dideoxy method (vide supra) using the plasmidvector and by the 7-DEAZA method (vide supra). The thus-determined cDNAsequence (about 500 bp) from the SP 6 promoter side (5' side of mRNA) ofpGEM4 is shown in FIG. 1.

In the cDNA sequence of the pGEM352-3 clone, there is a sequence codingfor the N-terminal ten amino acid residues of the purified human MACIFdescribed in section (1), with the translation initiation codon ATG at asite corresponding to amino acid residue No. -25 upstream from the Nterminus and the translation termination codon TAA at a sitecorresponding to the +104th amino acid residue downstream from the Nterminus. The open reading frame thus formed codes for a proteincomprising 128 amino acid residues. It is strongly suggested that theamino acid sequence corresponding to the region of -25 to -1 should bethe so-called secretion signal sequence very rich in hydrophobicity.

(5) Expression of human MACIF cDNA in oocytes and confirmation of itsbiological activities:

The above-mentioned pGEM352-3 clone was used for in vitro transcriptionof its cDNA portion into mRNA utilizing the SP6 promoter occurringupstream from the cDNA in the presence of SP6 RNA polymerase. Inparallel, the plasmid from a human IL-1α cDNA-containing clone was alsoused for in vitro transcription into mRNA. After microinjection of thosemRNAs together with TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA), asolvent therefor, into Xenopus laevis oocytes, in vitro translation waseffected by incubating the oocytes at 20° C. for 48 hours in modifiedBarth's medium [Gurdon, J. B., The Control of Gene Expression in AnimalDevelopment, Oxford University Press, (1974)] (with 0.1 μCi/μl of ³⁵S-Cys added in the case of immunoprecipitation). The oocytes were thendisrupted by sonication in a solution containing 0.01% Nonidet P40(NP40) (Sigma, U.S.A.) and centrifuged. The middle aqueous layer(hereinafter referred to as "translation product") was subjected toimmunoprecipitation and activity measurement.

1) Reactivity of translation products with antibodies(immunoprecipitation):

Each translation product was reacted with rabbit anti-human MACIFpolyclonal antibody or rabbit anti-human TNF polyclonal antibody, theantibody was bound to PANSORBIN (Staphylococcus aureus cells, Hoechst,West Germany), the binding mixture was centrifuged, the sediment waswashed three times with binding buffer (Affi-gel Protein A MAPS-II kit,Bio-Rad, U.S.A.) and then centrifuged with 0.17M glycine hydrochloride(pH 3.0), and the supernatant was measured for ³⁵ S-Cys using a liquidscintillation counter (TRI-CARB 460, Packard, U.S.A.). It was revealed,as shown below in Table 1, that only the translation product obtained bysubmitting pGEM352-3 to the in vitro transcription/translation systemcan react specifically with the rabbit anti-human MACIF polyclonalantibody.

                  TABLE 1                                                         ______________________________________                                               .sup.35 S-Cys Radioactivity (cpm)                                      RNA      Anti-Human MACIF                                                                             Anti-Human TNF                                        ______________________________________                                        TE       254            222                                                            235            208                                                   352-3    1577           381                                                            1249           278                                                   IL1-α                                                                            165            169                                                            218            410                                                   ______________________________________                                    

2) Biological activity of translation products:

2-1) Partial purification on an anti-human MACIF antibody column

The above translation products were adsorbed each on an antibody columnprepared by binding a purified mouse monoclonal anti-human MACIFantibody to activated Sepharose 4B (Pharmacia, Sweden). The column wasthen washed thoroughly with phosphate-buffered saline (PBS) containing0.1% NP40 and with 2M aqueous solution of sodium chloride containing0.1% NP40.

The translation product adsorbed on each antibody column was eluted with3M aqueous solution of sodium thiocyanate containing 0.1% NP40. Afterbuffer exchange for SGVB²⁺ containing 0.01% NP40, the eluate was usedfor activity measurement. The SGVB²⁺ buffer mentioned above has thefollowing composition: 0.1% gelatin, 5 mM sodium barbiturate buffer (pH7.4), 8.56% sucrose, 0.15 mM calcium chloride, 1 mM magnesium chloride.

2-2) Activity assay of translation products:

Human C5-C6 complex (stored frozen and thawed before use; C5,6^(f)) wasprepared from human C5 and C6 by the method of Dessauer et al.[Dessauer, A. et al., Acta Pathologica Microbiologica Scandinavia,Section C, Supplement 284, 92, 75-81 (1984)] and admixed with a guineapig erythrocyte suspension (10⁷ cells/ml), and the mixture was incubatedat 33° C. for 5 minutes. Then, after addition of C7, incubation wasperformed further for 15 minutes to give a guinea pig erythrocyte-humancomplement C5-7 complex (hereinafter referred to as "EC5-7intermediate"). The EC5-7 intermediate suspension (1.5×10⁸ /ml) wasadmixed with C8, C9 and a sample solution to make the total volume 1 ml(1.5×10⁷ /ml). Incubation was carried out at 37° C. for 1 hour. Inparallel, a control suspension prepared in the same manner but withoutaddition of any sample solution was incubated simultaneously. Thereaction mixture was centrifuged at 2,000×g for 5 minutes, thesupernatant was measured for absorbance (at 414 nm) for hemolysispercentage calculation. Thus, the number of sites (Z) per erythrocytewas calculated by the method of Hammer et al. [Hammer, C. H. et al.,Journal of Biological Chemistry, 256, 3995-4005 (1981)] and the ratio inpercentage of the Z value to the Z value for the control was taken as anindex of activity.

No activity was observed with TE and IL1-α, each used as a control,while significant antihemolytic activity was observed with human MACIF(FIG. 5). This activity was dose-dependent and was completelyneutralized with a mouse monoclonal antibody to human MACIF (FIG. 6).

It is therefore evident that the cDNA contained in the pGEM352-3 cloneobtained in the above described (4) codes for human MACIF.

(6) Construction of the expression vector pVY1:

The expression vector pVY1 was constructed as illustrated in FIG. 7.

1) The DNA of the vector pAdD26SV(A) No. 3 [obtained from Dr. H. Handa;made known by the paper: Kaufman, R. J. and Sharp, P. A., Molecular andCellular Biology, 2, 1304-1319 (1982)] was first cleaved with BglII,followed by phenol-chloroform extraction and ethanol precipitation. Theprecipitate thus obtained was dissolved in sterile distilled water,rendered blunt-ended in the conventional manner using Klenow enzyme(Boehringer Mannheim), then subjected to phenol-chloroform extractionand ethanol precipitation, and dissolved in sterile distilled water. Itwas further self-ligated using a DNA ligation kit (Takara Shuzo). Theligation mixture was used to transform competent cells of E. coli HB101.Plasmid DNAs were obtained from tetracycline-resistant transformants. Aportion of each of these DNAs was treated with BglII and electrophoresedon a 0.7% agarose gel. In this way, a clone having no BglII site anymore, pAdD26SV(A) No. 3(N), was obtained.

The plasmid DNA was then digested with EcoRI, followed byphenol-chloroform extraction and ethanol precipitation. The precipitatewas dissolved in sterile distilled water, and the EcoRI cleavage sitewas rendered blunt-ended using mung bean nuclease (Pharmacia), followedby phenol-chloroform extraction and ethanol precipitation. Theprecipitate thus obtained was dissolved in sterile distilled water.

2) The pKSV10 (Pharmacia) DNA was cleaved in the conventional mannerwith the restriction enzymes KpnI and BamHI and then renderedblunt-ended using T4 DNA polymerase (Takara Shuzo) and Klenow enzyme.After electrophoresis on a 0.7% agarose gel, the gel portion containinga fragment about 2.9 kbp in size was separated and the DNA was recoveredby electroelution.

3) The DNA fragment obtained as described in the above 1) and the DNAfragment obtained as described in the above 2) were ligated togetherusing a DNA ligation kit and the ligation mixture was used to transformcompetent cells of E. coli HB101.

Plasmid DNAs were prepared from tetracycline-resistant transformants bya conventional method. A part of each plasmid DNA was digested with PstIand the digest was subjected to 1.0% agarose gel electrophoresis. Inthis manner, the plasmid pVY1 giving bands at about 3.6 kbp, about 3.25kbp and about 1.5 kbp was obtained.

(7) Expression of human MACIF cDNA in CHO cells:

1) Construction of a human MACIF cDNA expression vector:

The plasmid pGEM352-3 was cleaved with the restriction enzymes SacI andHincII and a SacI/HincII DNA fragment of about 425 base pairs wasisolated and purified by agarose gel electrophoresis. This DNA fragmentwas rendered blunt-ended by treatment with mung bean nuclease.

pVY1 obtained as described in the above (6) was used as the expressionvector for use in CHO cells. Thus, pVY1 was cleaved with BglII andtreated with mung bean nuclease to render blunt the BglII cleavage end.The resulting blunt-ended DNA was ligated with the above-mentionedblunt-ended SacI/HincII DNA fragment using T4 DNA ligase and theligation mixture was used to transform E. coli HB101 to givetetracycline-resistant transformants. Plasmids were prepared from themby the alkaline bacteriolytic method [Birnboim, H. C. and Dolly, J.,Nucleic Acids Research, 7, 1513-1523 (1979)] and subjected torestriction enzyme analysis using PstI and so forth. In this way, aplasmid capable of allowing expression of the gene in question wasselected out.

2) Confirmation of the expression of the human MACIF gene in CHO cells:

The expression plasmid constructed in the above manner was transfectedinto DHFR-deficient CHO cells [Urlaub, G. and Chasin, L. A., Proc. Natl.Acad. Sci. U.S.A., 77, 4216-4220 (1980)] by the calcium phosphate method(vide supra). A transformant growing on a selective medium [MEM alpha(-), Gibco] was obtained.

The transformant CHO cell strain was grown on the selective medium and5×10⁵ cells thereof were washed three times with phosphate-bufferedsaline (PBS) and suspended in 500 μl of the culture supernatant obtainedby cultivating hybridoma cells capable of producing a monoclonalantibody (IgG1) to human MACIF. The reaction was allowed to proceed for1 hour with ice cooling. In parallel, a control run was carried out inthe same manner using the culture supernatant resulting from cultivationof hybridoma cells capable of producing a monoclonal antibody (IgG1) tohuman protein S. After reaction, the cells were washed three times withPBS containing 2% fetal calf serum and 0.1% sodium azide (hereinafterreferred to as "washing solution"), and then allowed to react, in aconcentration of 1×10⁶ cells/ml, with FITC (fluoresceinisothiocyanate)-labeled anti-mouse immunoglobulin (Amersham Japan)50-fold diluted with the same washing solution, for 30 minutes with icecooling. The cells were then washed three times with the washingsolution and analyzed with a flow cytometer (EPICS PROFILE, Coulter,U.S.A.). When the anti-human MACIF antibody was used, the fluorescenceintensity was found shifted to a higher level, as shown in FIG. 8a andFIG. 8b, indicating the binding of the anti-human MACIF antibody to thecells. It was thus confirmed that the transformed CHO cells hadexpressed human MACIF.

Furthermore, the transformed CHO cells were washed with PBS, thenincubated with phosphatidylinositol-specific phospholipase C (PI-PLC,Funakoshi, Sapporo Breweries) at 37° C. for 30 minutes, washed threetimes with the washing solution and subjected to fluorescent stainingwith the anti-human MACIF monoclonal antibody and with the FITC-labelledanti-mouse immunoglobulin in the same manner as above. As seen in FIG.8c, the fluorescence intensity lowered to the control level upontreatment with PI-PLC, indicating the release of human MACIF expressedin the transformed CHO cells from the membrane upon treatment withPI-PLC. It was thus confirmed that the human MACIF expressed on thetransformed CHO cells had been expressed on the cell membrane as a PIanchored protein susceptible to cleavage with PI-PLC.

3) Biological activity evaluation of the protein resulting from theexpression of human MACIF cDNA in CHO cells:

For confirming the biological activity feature of human MACIF expressedin the transformed CHO cells mentioned above, transformant CHO cell wereprepared in large quantities by cultivation thereof, washed with PBS andsolubilized by treating the cells overnight at 4° C. with NP40 at 2% inPBS containing various proteinase inhibitors [1 mM benzamidine, 2 mMphenylmethylsulfonyl fluoride (PMSF), 2 mM ethylenediaminetetraaceticacid (EDTA) and 2 mM ethylene glycol bis(β-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), all obtained from Sigma]. Eachsupernatant obtained by centrifugation (12,000 rpm, 30 minutes) waspartially purified by the method mentioned hereinabove under (5)-2-1)using an anti-human MACIF monoclonal antibody column and then subjectedto biological activity evaluation.

The MAC formation inhibiting activity (reactive lysis inhibitingactivity) of the human MACIF partially purified from the transformantCHO cells was evaluated by the method described under (5)-2-2). As shownin FIG. 9, the human MACIF expressed in the CHO cells exhibited MACformation inhibiting activity, which was dose-dependent. This inhibitoryactivity was neutralized by a mouse monoclonal antibody to humanerythrocyte-derived MACIF.

The above results clearly indicate that the human MACIF expressed in CHOcells is equivalent in activity to human erythrocyte-derived MACIF.

(8) Expression in Escherichia coli of a gene coding for a modified humanMACIF protein

1) Construction of a modified human MACIF protein expression vector andexpression in Escherichia coli

1-1) Preparation of a vector for expression in Escherichia coli

The vector pYEJ001 (Pharmacia; FIG. 10) for expression in Escherichiacoli was cleaved with the restriction enzyme HindIII, and the vectormain body containing the origin of replication and the promoter forexpression of the gene in question was isolated and purified by agarosegel electrophoresis and then treated with alkaline phosphatase forterminal phosphate group elimination.

The DNA fragment to be inserted into the vector, inclusive of the SDsequence, modified human MACIF protein gene and transcriptionterminator, was prepared in the following manner.

1-2) Preparation of an insert DNA fragment for the expression of amodified human MACIF protein (E103) comprising the peptide sequence upto the 103rd amino acid residue

pGEM352-3 was cleaved with the restriction enzymes PstI, HincII andBamHI and a DNA fragment of about 310 base pairs was isolated andpurified by agarose gel electrophoresis. Separately, the synthetic DNAsB and C shown by the following formula (VI) were terminallyphosphorylated using polynucleotide kinase. ##STR12##

The synthetic DNA A (shown above) and the phosphorylated B were heatedto 90° C. and then slowly cooled down to 16° C. for annealing. Thephosphorylated C and the synthetic DNA D (shown above) were treated inthe same manner for annealing. The two DNA annealing products and thepreviously prepared DNA of about 310 base pairs were ligated togetherusing T4 DNA ligase, and a DNA fragment of about 375 base pairscontaining the region coding for the modified human MACIF protein E103was isolated and purified by agarose gel electrophoresis.

1-3) Preparation of an insert DNA fragment for the expression of amodified human MACIF protein (E86) comprising the peptide sequence up tothe 86th amino acid residue

The previously prepared DNA fragment of about 310 base pairs was cleavedwith the restriction enzyme MboII and a DNA fragment of about 250 basepairs was isolated and purified by agarose gel electrophoresis.

Separately, two (B and C) of the four synthetic DNAs of the formulas(VII): ##STR13## were terminally phosphorylated using polynucleotidekinase. A and the phosphorylated B were heated to 90° C. and thengradually cooled to 16° C. for effecting annealing. The phosphorylated Cand unphosphorylated D were treated in the same manner for annealing.The two annealing product DNAs and the previously prepared DNA fragmentof about 250 base pairs were ligated together using T4 DNA ligase and aDNA fragment of about 325 base pairs containing the region coding forthe modified MACIF protein E86 was isolated and purified by agarose gelelectrophoresis.

1-4) Preparation of a vector for the expression of the modified humanMACIF protein in Escherichia coli and transformation therewith:

The thus-prepared two DNA fragments were terminally phosphorylated usingpolynucleotide kinase and then ligated with the separately preparedvector main body using T4 DNA ligase. The ligation mixture was used totransform E. coli K12JM109. Plasmids were prepared from thetransformants obtained by the alkaline method (vide supra) and analyzedusing restriction enzymes (e.g. by cleavage with PstI), and a plasmidwith the gene in question inserted therein in the direction enabling thegene to be expressed was selected out.

1-5) Expression and cultivation:

One volume of 2×YT medium (16 g of Bactotryptone, 10 g of yeast extractand 5 g of sodium chloride per liter) supplemented with 100 μg/ml ofampicillin was inoculated with 1/50 volume of a preculture of arecombinant E. coli strain allowing expression of the gene in question.Then, culture was shaken at 37° C. and the recombinant E. coli strainwas grown until cell density had reached about 5×10⁷ /ml, and thenisopropyl thiogalactopyranoside was added in a concentration of 2.5 mM.After further 16 hours of cultivation, cells were harvested.

The cells collected were suspended in PBS containing 2 mM EDTA, 2 mMEGTA, 1 mM benzamidine, 2 mM phenylmethylsulfonyl fluoride (PMSF) (allpurchased from Sigma) and disrupted using a Manton-Gaulin homogenizer.

2) Purification of the protein resulting from the expression of themodified human MACIF protein gene in Escherichia coli and biologicalactivity assay:

2-1) Purification:

The Escherichia coli cell disruption product prepared as described abovewas centrifuged (12,000 rpm, 30 minutes) and the sediment was stirredovernight at 4° C. in the presence of 6M guanidine hydrochloride forsolubilization.

The remaining insoluble matter was removed by centrifugation under thesame conditions as mentioned above. The expression product protein (E103or E86) contained in the supernatant was reconstituted by treatment inthe presence of oxidized-form and reduced-form glutathione (Sigma)according to the method of Winkler and Blaber [Winkler, M. E. andBlaber, M., Biochemistry, 256, 4041-4045 (1986)].

The reconstitution mixture was thoroughly dialyzed against 10 mM Trisbuffer (pH 8.0) and then purified by application to Q-Sepharose(Pharmacia) preequilibrated with the same buffer.

2-2) Activity of the modified human MACIF protein (E103):

As shown in FIG. 11, the reactive lysis inhibiting activity dataobtained by the method described in section (5)-2-2) showed two peaks.However, the second activity peak (around fraction No. 25) alone waspartially neutralized by the anti-human MACIF monoclonal antibody.

As shown in FIG. 12, polyacrylamide gel electrophoresis performed in thepresence of sodium dodecyl sulfate (SDS-PAGE) gave a main band in accordwith the second activity peak. Therefore, these activity fractions werefurther purified on anti-human MACIF antibody column and reactive lysisinhibiting activity was measured in the same manner. As shown in FIG.13, the purified expression product protein (E103) showed antihemolyticactivity in a dose-dependent manner and the inhibitory activity wascompletely neutralized by the anti-human MACIF monoclonal antibody.

2-3) Activity of the modified human MACIF protein (E86):

Unlike the case of E103, the activity data obtained failed to show amain peak of activity neutralizable by the antibody (FIG. 14). However,based on the results of SDS-PAGE analysis as shown in FIG. 15, the mainband-containing fractions (Nos. 26-30) were purified on an anti-humanMACIF antibody column, followed by reactive lysis inhibiting activitymeasurement. As shown in FIG. 16, the purified expression productprotein (E86) showed antihemolytic activity in a dose-dependent manner.Furthermore, the inhibitory activity was completely neutralized by theantibody.

The above results indicate that the modified human MACIF proteinexpressed in Escherichia coli has the same activity as that of humanerythrocyte-derived MACIF and that human MACIF can exhibit reactivelysis inhibiting activity even when it has no carbohydrate chain or noPI anchor.

(9) Expression of the gene coding for a modified human MACIF protein inCHO cells and confirmation of its biological activity:

1) Expression of a modified human MACIF protein (C86) in CHO cells:

1-1) Construction of a recombinant plasmid for expression in CHO cells:

The plasmid pVY1 obtained in section (6) was used as the vector forexpression in CHO cells. Thus, pVY1 was cleaved with BglII and submittedto the following recombinant plasmid construction.

pGEM352-3 was cleaved with Ecg0109I and StyI and ligated with asynthetic DNA of the formula (VIII) phosphorylated at the StyI cleavagesite and a synthetic DNA of the formula (IX) phosphorylated at theEco0109I cleavage site, using T4 DNA ligase. ##STR14##

A DNA fragment of 341 base pairs was then isolated and purified byagarose gel electrophoresis. This DNA fragment was ligated with theBglII-cleaved pVY1 using T4 DNA ligase and the ligation mixture was usedto transform E. coli HB101 to give tetracycline-resistant transformants.Plasmids were prepared therefrom by the alkaline bacteriolytic methodand analyzed using restriction enzymes (PstI etc.) and a recombinantexpression plasmid for the gene in question was selected out.

1-2) Confirmation of gene expression in CHO cells:

DHFR-deficient CHO cells (vide supra) were transfected with theexpression plasmid constructed in the above manner by the calciumphosphate method. A transformant strain capable of growing in aselective medium [MEM alpha (-), Gibco] in the presence of methotrexatewas obtained and used in the subsequent studies.

10⁸ CHO cells transformed in the above manner were cultured and theculture supernatant was subjected to affinity chromatography using ananti-MACIF monoclonal antibody column and following the proceduredescribed in section (5)-2-1). Fractions bound to this antibody columnwere eluted with 3M aqueous solution of sodium thiocyanate. Each eluatefraction was desalted on a PD-10 column (Pharmacia) and evaluated forthe presence or absence of antigen by competitive ELISA using a rabbitanti-naturally occurring human MACIF antibody. As a result, it wasconfirmed that a human MACIF antigen was present in a "bound" fractionin the above affinity chromatography.

This result proves that a modified human MACIF protein comprising thepeptide composed of the 1st to 86th amino acid residues had beensuccessfully expressed in CHO cells in accordance with the invention.

2) Expression of a modified human MACIF protein (C82) in CHO cells:

2-1) Construction of a recombinant plasmid for expression in CHO cells:

The procedure of (9)-1-1) was repeated except that a DNA fragment of theformula (X) phosphorylated at the Eco0109I site was used in expressionplasmid construction. ##STR15## 2-2) Confirmation of gene expression inCHO cells:

The procedure of (9)-1-2) was followed and a human MACIF antigen wasdetected in the culture supernatant obtained with CHO cells transformedwith the above-mentioned recombinant vector. This result indicated thata modified human MACIF protein comprising the peptide composed of the1st to 82nd amino acid residues had been successfully expressed in CHOcells in accordance with the present invention.

3) Expression of a modified human MACIF protein (C77) in CHO cells andconfirmation of its biological activity:

3-1) Construction of a recombinant plasmid for gene expression in CHOcells:

The plasmid pVY1 obtained as described in the above (6) was used as thevector for gene expression in CHO cells. pVY1 was cleaved with BglII andsubmitted to the following recombinant plasmid construction.

pGEM352-3 was cleaved with Eco0109I and StyI and ligated with asynthetic DNA fragment of the formula (VIII) phosphorylated at the StyIcleavage site and a synthetic DNA fragment of the formula (XI)phosphorylated at the Eco0109I cleavage site, using T4 DNA ligase.##STR16##

A DNA fragment of 314 base pairs was then isolated and purified byagarose gel electrophoresis. This DNA fragment was ligated with theBglII-cleaved pVY1 using T4 DNA ligase and the ligation mixture was usedto transform E. coli HB101 to give tetracycline-resistant transformants.Plasmids were prepared from these by the alkaline bacteriolytic methodand analyzed by using restriction enzymes (PstI etc.) and a recombinantplasmid capable of expression of the gene in question was selected out.

3-2) Confirmation of gene expression in CHO cells:

DHFR-deficient CHO cells (vide supra) were transfected with theexpression plasmid constructed in the above manner by the calciumphosphate method and a transformant capable of growing in a selectivemedium [MEM alpha (-) Gibco] in the presence of methotrexate wasisolated and used for further studies.

10⁸ CHO cells transformed by the above method were cultured and theculture supernatant was subjected to affinity chromatography using ananti-MACIF monoclonal antibody column and proceeding as described in(5)-2-1). Fractions bound to this antibody column were eluted with 3Maqueous solution of sodium thiocyanate and eluate fractions weredesalted on a PD-10 column (Pharmacia) and assayed for the presence orabsence of antigen by competitive ELISA using a rabbit anti-MACIFantibody. The presence of a MACIF antigen was confirmed in a "bound"fraction in the above affinity chromatography.

This result indicates that a modified human MACIF protein comprising thepeptide composed of the 1st to 77th amino acid residues had beenexpressed successfully in CHO cells.

3-3) Confirmation of biological activity:

A sample of the modified human MACIF protein C77 as partially purifiedfrom the culture supernatant resulting from mass culture of thetransformed CHO cells by the purification procedure described in theabove (9)-3-2) was subjected to buffer exchange for SGVB²⁺, followed byMAC formation inhibiting activity assay, which was performed asdescribed in (5)-2-2). As shown in FIG. 17, the modified human MACIFprotein C77 expressed in CHO cells showed MAC formation inhibitingactivity in a dose-dependent manner. The inhibitory activity wasneutralized by a mouse monoclonal antibody to erythrocyte-derived,naturally occurring human MACIF.

From the above results, it is evident that the modified human MACIFprotein C77 expressed in CHO cells and secreted into medium has the sameactivity as that of human erythrocyte-derived, naturally occurringMACIF.

4) Expression of a modified human MACIF protein (C76) in CHO cells andconfirmation of its biological activity:

4-1) Construction of a recombinant plasmid for gene expression in CHOcells:

The procedure was the same as that described in (9)-1-1) except that aDNA fragment of the formula (XII) phosphorylated at the Eco0109I sitewas used in expression plasmid construction. ##STR17## 4-2) Confirmationof gene expression in CHO cells:

By following the procedure of (9)-1-2, a human MACIF antigen wasdetected in the culture supernatant resulting from cultivation of cellstransformed with the above recombinant vector. This result indicatesthat a modified human MACIF protein comprising the peptide composed ofthe 1st to 76th amino acid residues had successfully been expressed inCHO cells.

4-3) Confirmation of biological activity:

The culture supernatant resulting from mass culture of the transformedCHO cells was used for evaluating the MAC formation inhibiting activityof the modified human MACIF protein (C76) expressed in CHO cells byfollowing the procedure described in (9)-3-3). As shown in FIG. 18, C76showed the activity in a dose-dependent manner.

From the results mentioned above, it is evident that the modified humanMACIF protein (C76) expressed in CHO cells and secreted into medium hasthe same activity as that of human erythrocyte-derived, naturallyoccurring MACIF.

5) Expression of a modified human MACIF protein (C75) in CHO cells:

5-1) Construction of a recombinant plasmid for gene expression in CHOcells:

The procedure was the same as that described in (9)-1-1) except that aDNA fragment of the formula (XIII) phosphorylated at the Eco0109I sitewas used in expression plasmid construction. ##STR18## 5-2) Confirmationof gene expression in CHO cells:

By following the procedure of (9)-1-2), a human MACIF antigen wasdetected in the culture supernatant obtained with cells transformed withthe above-mentioned recombinant vector. This result indicates that amodified human MACIF protein comprising the peptide composed of the 1stto 75th amino acid residues had been successfully expressed in CHOcells.

6) Expression of a modified human MACIF protein (C70) in CHO cells andconfirmation of its biological activity:

6-1) Construction of a recombinant plasmid for gene expression in CHOcells:

The procedure was the same as that described in (9)-1-1) except that aDNA fragment of the formula (XIV) phosphorylated at the Eco0109I sitewas used in expression plasmid construction. ##STR19## 6-2) Confirmationof gene expression in CHO cells:

A human MACIF antigen was detected, by the procedure of (9)-1-2), in theculture supernatant obtained with cells transformed with the aboverecombinant vector. This result indicates that a modified human MACIFprotein comprising the peptide composed of the 1st to 70th amino acidresidues had been expressed successfully in CHO cells.

6-3) Confirmation of biological activity:

The MAC formation inhibiting activity of the modified human MACIFprotein (C70) expressed in CHO cells was measured using the culturesupernatant obtained by mass culture of the transformed CHO cells andfollowing the procedure of (9)-3-3). As shown in FIG. 19, C70 showed theactivity in a dose-dependent manner.

From the results mentioned above, it is evident that the modified humanMACIF protein (C70) expressed in CHO cells and secreted into medium hasthe same activity as that of human erythrocyte-derived, naturallyoccurring MACIF.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

What is claimed is:
 1. A fusion protein consisting ofan N-terminal MACIF(Membrane Attack Complex Inhibition Factor) moiety having the followingsequence: ##STR20## wherein X is H or Met and a C-terminal moietyconsisting of a heterologous phosphatidylinosirol (PI) anchor domain,wherein a PI membrane anchor is attached to said domain.